The four serotypes of dengue viruses (DENV1-4; Flavivirus; Flaviviridae) are the most important mosquito- borne arboviruses infecting humans. In tropical regions of the world, DENV are hyper-endemic with approximately 2.5 billion people at risk for epidemic transmission. The principal vector of DENV is Aedes aegypti. Currently, DENV control efforts rely primarily on insecticide applications, since vaccines or antiviral drugs are not available for widespread use. Additionally, insecticide resistance is increasing among Ae. aegypti populations, requiring the development of alternative, genetic vector/virus control strategies. One such novel strategy is population replacement in which wild-type mosquitoes susceptible for DENV are replaced by genetically-modified, virus-resistant mosquitoes. The purpose of this grant application is to generate transgenic Ae. aegypti lines, which are refractory to all four serotypes of DENV. The mosquito acquires DENV from a human host by intake of a viremic bloodmeal. The midgut epithelium is the first mosquito tissue that becomes infected with DENV. In the midgut, DENV needs to establish infection foci before being able to disseminate to secondary tissues including the salivary glands, which have to be infected before the virus can be transmitted to a new human host. The RNA interference (RNAi) pathway in Ae. aegypti is the major antiviral immune pathway targeting arboviruses. We have shown that triggering RNAi against DENV2 in midgut tissue completely eliminates infection before the virus can establish infection foci. We recently generated transgenic Ae. aegypti that express an inverted-repeat (IR) dsRNA derived from the genome of DENV2 in midgut tissue of bloodfed mosquitoes as infection begins. One DENV2-refractory transgenic line (Carb109) has been maintained for more than 30 generations and the Carb109 transgene has been introgressed into wild-type Ae. aegypti converting these mosquitoes from a highly susceptible DENV2 phenotype to a completely refractory phenotype. Here, we propose to engineer RNAi-mediated resistance in Ae. aegypti to all four DENV serotypes using a novel design for the IR effector constructs. We will design 190 bp IR constructs each targeting a highly conserved region of viral RNA encoding DENV1-4 NS5. The antiviral effector genes will be critically important components for a population replacement-based control strategy of DENV in the field. The proposed research effort will further strengthen the concept of population replacement and complement ongoing studies by other research groups to move anti-viral effector constructs into wild-type mosquito populations.